Rabbit (polyclonal)
Anti-c-Ret [pY1016] Phosphospecific
Antibody, Unconjugated
PRODUCT ANALYSIS SHEET
Catalog Number:
44-277G (10 mini-blot size)
Lot Number:
See product label
Volume:
100 μL
Form of Antibody:
Rabbit polyclonal immunoglobulin in Dulbecco’s phosphate buffered saline (without Mg2+ and Ca2+),
pH 7.3 (+/- 0.1), 50% glycerol with 1.0 mg/mL BSA (IgG, protease free) as a carrier.
Preservative:
0.05% sodium azide (Caution: sodium azide is a poisonous and hazardous substance. Handle with care
and dispose of properly.)
Purification:
Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been
negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to
remove antibody that is reactive with non-phosphorylated c-Ret. The final product is generated by
affinity chromatography using c-Ret-derived peptide that is phosphorylated at tyrosine 1016.
Immunogen:
The antiserum was produced against a chemically synthesized phosphopeptide derived from the region
of mouse c-Ret that contains tyrosine 1016.
Target Summary:
c-Ret is a 170 kDa tyrosine kinase receptor for glial cell line-derived neurotrophic factors (GDNFs:
neurturin, persephin, and artemin) and plays an important role in the development of the enteric
nervous system and the kidney. In humans, inactivating mutations of c-Ret result in Hirschsprung's
disease, and constitutively active c-Ret mutations result in endocrine neoplasia and familial medullary
thyroid carcinoma. Activation of the c-Ret can initiate a cascade of events including the activation of
the Ras → Raf → ERK1&2 pro-growth signaling pathway. c-Ret is regulated by phosphorylation on
multiple tyrosine and serine sites. Phosphorylation of c-Ret on serine 696 by protein kinase A (PKA)
promotes lamellipodia formation of neuronal cells induced by GDNF, a critical step in neuritegenesis.
Reactivity:
Human. Mouse c-Ret has not been tested but is expected to react (100% homology).
Applications:
The antibody has been used in Western blotting and Immunostaining.
Suggested Working
Dilutions:
For Western blotting applications, we recommend using the antibody at a 1:1000 starting dilution. The
optimal antibody concentration should be determined empirically for each specific application.
Storage:
Store at −20oC. We recommend a brief centrifugation before opening to settle vial contents. Then,
apportion into working aliquots and store at −20oC. For shipment or short-term storage (up to one
week), 2-8oC is sufficient.
Expiration Date:
Expires one year from date of receipt when stored as instructed.
Positive Controls
Used:
GDNF-stimulated Neuro-2A cells or TT medullary thyroid carcinoma cells.
This product is for research use only. Not for use in diagnostic procedures.
www.invitrogen.com
Invitrogen Corporation • 542 Flynn Rd • Camarillo • CA 93012 • Tel: 800.955.6288 • E-mail: techsupport@invitrogen.com
This antibody is manufactured under a licensed process covered by Patent # 5, 599, 681.
PI44277G
(Rev 11/08) DCC-08-1089
Important Licensing Information - These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or our website, www.invitrogen.com). By
use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not
intended for human or animal diagnostic, therapeutic or commercial use.
FAK [pY397], Cat. # 44-625G
FAK [pY861], Cat. # 44- 626G
β-Catenin [pS45], Cat. # 44-208G
Rac/Cdc42 [pS71], Cat. # 44-214G
SHP2 [pY580], Cat. # 44-558G
SHP2 [pS576], Cat. # 44-557G
STAT1 [pY701], Cat. # 44-377G
Related Products:
Antibodies:
c-Ret [pS696], Cat. # 44-274
ERK1/2[pTpY185/187], Cat. # 44-680G
PAK1/2/3 [pT423], Cat. # 44-942G
PAK 1/2/3 [pS141], Cat. # 44-940G
Akt [pS473], Cat. # 44-622G
Akt [pT308], Cat. # 44-602G
References:
Iervolino, A., et al. (2006) The receptor-type protein tyrosine phosphatase J antagonizes the
biochemical and biological effects of RET-derived oncoproteins. Cancer Res. 66(12):6280-6287.
Zhang, J. and E.J. Huang (2006) Dynamic expression of neurotrophic factor receptors in postnatal
spinal motoneurons and in mouse model of ALS. J. Neurobiol. 66(8):882-895.
Carlomagno, F., et al. (2006) BAY 43-9006 inhibition of oncogenic RET mutants. J. Nat’l. Cancer
Inst. 98(5):326-334.
Panta, G.R., et al. (2004) RET signals through focal adhesion kinase in medullary thyroid cancer cells.
Surgery 136(6):1212-1217.
Fukuda, T., et al. (2002) Novel mechanism of regulation of Rac activity and lamellipodia formation by
RET tyrosine kinase. J. Biol. Chem. 277(21):19114-19121.
Carlomagno, F., et al. (2002) The kinase inhibitor PP1 blocks tumorigenesis induced by RET
oncogenes. Cancer Res. 62(4):1077-1082.
Cohen, M.S., et al. (2002) Inhibition of medullary thyroid carcinoma cell proliferation and RET
phosphorylation by tyrosine kinase inhibitors. Surgery 132(6):960-967.
You, L., et al. (2001) Glial cell-derived neurotrophic factor (GDNF)-induced migration and signal
transduction in corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 42(11):2496-2504.
Jing, S., et al. (1996) GDNF-induced activation of the Ret protein tyrosine kinase is mediated by
GDNFR-α, a novel receptor for GDNF. Cell 85(7):1113-1124.
Edery, P., et al. (1994) Mutations of the RET proto-oncogene in Hirschsprung’s disease. Nature
367(6461):378–380.
Peptide Competition and Mutant Analysis
Lysates prepared from Neuro-2A cells stimulated with GDNF (1-5) were
resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to
PVDF. Membranes were either left untreated (1-4) or treated with lambda
(λ) phosphatase (5), blocked with a 3% BSA-TBST buffer overnight at 4oC
and incubated with c-Ret [pY1016] antibody for two hours at room
temperature in a 3% BSA-TBST buffer, following prior incubation with: no
peptide (1&5), the non-phosphopeptide corresponding to the immunogen
(2), a generic phosphotyrosine containing peptide (3), or, the
phosphopeptide immunogen (4). After washing, membranes were incubated
with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase (cat.# ALI4405) and
signals were detected using Pierce SuperSignal™ detection reagent.
The data show that only the peptide corresponding to c-Ret [pY1016] blocks
the antibody signal and that phosphatase stripping eliminates the signal,
thereby demonstrating the phospho- specificity of the antibody.
This product is for research use only. Not for use in diagnostic procedures.
www.invitrogen.com
Invitrogen Corporation • 542 Flynn Rd • Camarillo • CA 93012 • Tel: 800.955.6288 • E-mail: techsupport@invitrogen.com
This antibody is manufactured under a licensed process covered by Patent # 5, 599, 681.
PI44277G
(Rev 11/08) DCC-08-1089
Important Licensing Information - These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or our website, www.invitrogen.com). By
use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not
intended for human or animal diagnostic, therapeutic or commercial use.
Western Blotting Procedure
1. Lyse approximately 107 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer,
is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. This cell
lysis buffer formulation is available as a separate product which requires supplementation with protease inhibitors immediately prior to
use (Cat. # FNN0011). Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible
with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific
application.
2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifuged
at 100,000 x g for 30 minutes for greater clarification.
3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the
Bradford assay. Polypropylene tubes are recommended for storing cell lysates.
4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS,
0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100oC.
5. Load 10-30 μg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by
SDS-PAGE.
6. In preparation for the Western transfer, cut a piece of PVDF slightly larger than the gel. Soak the membrane in methanol for 1 minute,
then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used.
7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below)
for 2 minutes.
8. Assemble the gel and membrane into the sandwich apparatus.
9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature.
10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes.
11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4oC.
12. Incubate the blocked blot with primary antibody at a 1:1000 starting dilution in Tris buffered saline supplemented with 3% Ig-free BSA
and 0.1% Tween-20 overnight at 4oC or for two hours at room temperature.
13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20.
14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab’)2 anti-rabbit IgG alkaline phosphatase conjugate
(Cat. # ALI4405) or goat F(ab’)2 anti-rabbit IgG horseradish peroxidase conjugate (Cat. # ALI4404) in conjunction with your
chemiluminescence reagents and instrumentation.
Cell Lysis Buffer
Formulation:
10 mM Tris, pH 7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1 mM NaF
20 mM Na4P2O7
2 mM Na3VO4
0.1% SDS
0.5% sodium deoxycholate
1% Triton-X 100
10% glycerol
1 mM PMSF (made from a
0.3 M stock in DMSO)
or 1 mM AEBSF (water
soluble version of PMSF)
60 μg/mL aprotinin
10 μg/mL leupeptin
1 μg/mL pepstatin
(alternatively, protease inhibitor cocktail
such as Sigma Cat. # P2714 may be used)
Transfer Buffer
Formulation:
2.4 gm Tris base
14.2 gm glycine
200 mL methanol
Q.S. to 1 liter, then add
1 mL 10% SDS.
Cool to 4oC prior to use.
Tris Buffered Saline
Formulation:
20 mM Tris-HCl, pH 7.4
0.9% NaCl
Blocking Buffer
Formulation:
100 mL Tris buffered saline
3 gm Ig-free BSA
0.1 mL Tween 20
This product is for research use only. Not for use in diagnostic procedures.
www.invitrogen.com
Invitrogen Corporation • 542 Flynn Rd • Camarillo • CA 93012 • Tel: 800.955.6288 • E-mail: techsupport@invitrogen.com
This antibody is manufactured under a licensed process covered by Patent # 5, 599, 681.
PI44277G
(Rev 11/08) DCC-08-1089
Important Licensing Information - These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or our website, www.invitrogen.com). By
use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not
intended for human or animal diagnostic, therapeutic or commercial use.
Peptide Competition Experiment
Invitrogen’s Phosphorylation Site Specific Antibodies (PSSAs) have been developed to enable the specific and sensitive detection of
phosphorylation of particular amino acid residues in target proteins, while circumventing the need for protein purification, phosphopeptide
mapping or handling radioactivity. The specificity of a PSSA in each experimental system can be confirmed through peptide competition. In this
technique, aliquots of antibody are pre-incubated with peptide containing the sequence of the phosphopeptide immunogen used to raise the
PSSA and the corresponding non-phosphopeptide. Following preincubation with the peptide, each antibody preparation is then used as a probe
in antibody-based detection methods, such as Western blotting, immunocytochemistry, flow cytometry, or ELISA. With a PSSA specific for the
phosphorylated target protein, pre-incubation with an excess of peptide containing the sequence of the phosphopeptide immunogen will block
all antigen binding sites, while pre-incubation with the corresponding non-phosphopeptide will not affect the antibody.
Invitrogen has developed a line of control peptides specifically for use in peptide competition experiments with our PSSAs. These peptides,
available as separate Invitrogen catalog items, are provided in pairs which contain the sequences of the phosphopeptide immunogen and the
corresponding non-phosphopeptide.
In performing the Peptide Competition Experiment, it is important to note that the optimal dilutions of both antibody and peptide should be
determined empirically for each specific application. The optimal dilution of antibody in these procedures is below saturating, as determined by
previous experiments in your system. If an optimal antibody dilution has not been determined in your system, please refer to the Suggested
Working Dilution on the antibody Product Analysis Sheet for guidance on an appropriate starting dilution. The optimal dilution of peptide used
in these procedures will depend on the overall affinity or avidity of the antibody, as well as the quantity of the target antigen. A 50-150 fold
molar excess of peptide to antibody is found to be effective for most peptide competition experiments.
In the example presented below, the PSSA is used as a dilution of 1:1000 and the peptides are used at a concentration of 333 nM. The total
volume of the phosphopeptide and non-phosphopeptide-pre-incubated antibody preparations is 2 mL, sufficient for probing Western blot strips,
as well as for use in other antibody-based detection methods. Under these conditions, the molar excess of peptide to antibody is ≥50.
Procedure:
1. Prepare three identical test samples, such as identical PVDF or nitrocellulose strips to which the protein of interest has been transferred.
The test samples should be blocked using a blocking buffer, such as Tris buffered saline supplemented with 0.1% Tween 20, and either 5%
BSA or 5% non-fat dried milk.
2. Prepare 6.5 mL of working antibody stock solution (1:1000 in this example) by adding 6.5 μL of antibody stock solution to 6.5 mL of
buffer containing blocking protein, such as TBS supplemented with 0.1% Tween 20, and either 3% BSA or 3% non-fat dried milk.
3. Apportion the unused PSSA into working aliquots and store at −20°C for future use (the stock PSSA contains 50% glycerol and will not
freeze at this temperature).
4. Allow the lyophilized control peptides to reach room temperature, ideally under desiccation.
5. Reconstitute each of the control peptides (supplied at 0.1 mg/vial) to a concentration of 66.7 μM with nanopure water. For a peptide with a
molecular mass of 1500 (stated on the peptide Product Analysis Sheet), reconstitution with 1 mL water yields a solution with a
concentration of 66.7 μM.
6. Apportion the unused reconstituted peptide solutions into working aliquots and store at -20°C for future use.
7. Label 3 test tubes as follows:
•
tube 1: water only no peptide control
•
tube 2: phosphopeptide
•
tube 3: non-phosphopeptide
8. Into each tube, pipette the following components
•
tube 1: 2 mL diluted PSSA solution plus 10 μL nanopure water
•
tube 2: 2 mL diluted PSSA solution plus 10 μL phosphopeptide
•
tube 3: 2 mL diluted PSSA solution plus 10 μL non-phosphopeptide
9. Incubate the three tubes for 30 minutes at room temperature with gentle rocking. During this incubation, the peptides have the chance to
bind to the combining site of the antibody.
10. At the end of the incubation step, transfer the contents of each of the three tubes to clean reaction vessels containing one of the three
identical test samples.
For Western blotting strips:
•
Incubate the strips with the pre-incubated antibody preparations for 1 hour at room temperature or overnight at 4°C.
•
Wash each strip four times, five minutes each, to remove unbound antibody.
•
Transfer each strip to a new solution containing a labeled secondary antibody [e.g., goat F(ab’)2 anti-rabbit IgG alkaline phosphatase
conjugate (Cat. # ALI4405) or goat F(ab’)2 anti-rabbit IgG horseradish peroxidase conjugate (Cat. # ALI4404)].
•
Remove unbound secondary antibody by thorough washing, and develop the signal using your chemiluminescent reagents and
instrumentation.
The signal obtained with antibody incubated with the “Water Only, No Peptide Control” (Tube 1), represents the maximum signal in the assay.
This signal should be eliminated by pre-incubation with the “Phosphopeptide” (Tube 2), while pre-incubation with the “Non-Phosphopeptide”
(Tube 3) should not impact the signal. If the “Phosphopeptide” only partially eliminates the signal, repeat the procedure using twice the volume
of water or peptide solutions listed in Step 8. If partial competition is seen following pre-incubation with the “Non-Phosphopeptide”, repeat the
procedure using half the volumes of water or peptide solutions listed in Step 8.
This product is for research use only. Not for use in diagnostic procedures.
www.invitrogen.com
Invitrogen Corporation • 542 Flynn Rd • Camarillo • CA 93012 • Tel: 800.955.6288 • E-mail: techsupport@invitrogen.com
This antibody is manufactured under a licensed process covered by Patent # 5, 599, 681.
PI44277G
(Rev 11/08) DCC-08-1089
Important Licensing Information - These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or our website, www.invitrogen.com). By
use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not
intended for human or animal diagnostic, therapeutic or commercial use.